Systematic evolution of ligands by exponential enrichment (SELEX) has been used to select one or more single-stranded nucleic acids which specifically bind a target (Tuerk & Gold, Science 249:505-510, 1990; Ellington & Szostak, Nature 346:818-822, 1990). Such nucleic acids are called aptamers. An aptamer, which antagonizes the activity of vascular endothelial growth factor (VEGF), is used to treat age-related macular degeneration.
U.S. Pat. No. 5,270,163 describes the SELEX method. A library of nucleic acids is contacted with a target, and those nucleic acids specifically bound to the target are partitioned from the remainder of nucleic acids in the library which do not specifically bind the target. The partitioned nucleic acids are amplified to yield a ligand-enriched pool. Multiple cycles of binding, partitioning, and amplifying (i.e., selection) result in identification of one or more aptamers with the desired activity. The typical library is constructed with many nucleic acids of different sequences that confer structurally diverse conformations so selection of the desired nucleic acid(s), which are present at low frequency in the library, can be difficult.
U.S. Pat. No. 6,376,190 describes increasing or decreasing frequency of nucleic acids in a library by their binding to a chemically synthesized peptide. This method requires a purified or recombinant (i.e., non-native) target for binding to members of the library. It teaches away from enriching for particular members of a library using a complex mixture of proteins in more than two rounds. See columns 9 to 16.
It would be convenient if a library containing candidate aptamers could be focused against a proteome (i.e., complex mixture of native biomolecules) over multiple rounds of binding, partitioning, and amplifying. In accordance with the present invention, focusing increases the fraction of nucleic acids binding to the native biomolecules (e.g., randomized sequences which are present in substantially equal abundance tend to frequencies reflecting strength of target binding and abundance of targets in the proteome) and decreases the fraction of nucleic acids in the library which do not bind to the proteome. Sequence complexity of the library (i.e., number of different nucleic acids according to their sequences) and the size of library (i.e., total number of nucleic acids) are reduced by focusing. Individual members that would otherwise tend to predominant the focused library because of strong target binding and high target abundance in the proteome may mask the relatively less abundant (i.e., scarce) candidate aptamers in the library and prevent their isolation. In accordance with the present invention, frequently isolated members of a library may be depleted therefrom as they are identified by their sequences.
Therefore, it is an objective(s) of the invention to provide improved methods for SELEX, focused libraries of reduced complexity and enhanced relevancy to intended targets, easier and more efficient screening of focused libraries, or aptamers with desired biological activity other than target binding. Long-felt needs are addressed thereby.
The present invention is also directed to an improvement(s) in SELEX to construct focused libraries, deconvoluting libraries by one or more functional (i.e., non-binding) assays, increasing frequencies of one or more candidate aptamers which bind to native targets of a proteome, decreasing frequencies of one or more candidate aptamers which bind to native targets of a proteome, or any combination thereof that address the aforementioned problems. Methods for using and making these products (i.e., focused libraries, aptamers, and derivatives thereof) are provided. Further objectives and advantages of the invention are described below. Other advantages and improvements are described below or would be apparent from the disclosure herein.